Dimethyl succinate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dibasic esters

IUPAC Name:

Dibasic esters

Details on test material:

- Name of test material (as cited in study report): Dibasic esters (DBE)
- Physical state: Liquid
- Analytical purity: >99.5%
- Composition of test material, percentage of components: Dimethyl glutarate - 65.1%; dimethyl succinate - 17.8%; dimethyl adipate - 16.8%
- Stability under test conditions: Stable

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River Breding Laboratories, Kingston, NY, USA
- Age at study initiation: 4 weeks
- Weight at study initiation: 60 - 123 g
- Fasting period before study: Not applicable
- Housing: Paired, in stainless steel wire mesh cages
- Diet (e.g. ad libitum): Ad libiutum except during exposure
- Water (e.g. ad libitum): Ad libitum except during exposure
- Acclimation period: Approximately 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 deg C
- Humidity (%): 42 - 63%
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

5.6 µm

Remarks on MMAD:

MMAD / GSD: Aerosol particle size measured by cascade impactor and reported as mass median aerodynamic diameter and % of particles less than 10 micron aerodynamic diameter.
Mean particle size was 5.6 micron 72% of the generated aerosol was <10 micron
Chamber aerosol mass concentration determined by drawing known quantities of the chamber atmosphere through glass-fibre filters and calculating the filter weight gain per volume of atmosphere sampled.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: NYU style inhalation chamber
- Method of holding animals in test chamber: stainless steel "modules"
- Source and rate of air: Ambient air, 300L/minute
- Method of conditioning air: Not reported
- System of generating particulates/aerosols: nebuliser
- Temperature, humidity, pressure in air chamber: Measured but not reported
- Air flow rate: 300l/minute
- Air change rate: Not reported
- Method of particle size determination: Cascade impactor
- Treatment of exhaust air: Not reported

TEST ATMOSPHERE
- Brief description of analytical method used: Mass concentration determined gravimetrically following trapping on glass fibre filters. Identity by GC analysis of solvent traps collecting both aerosol and vapour
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Justification for use and choice of vehicle: None used

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were analysed by gas chromatography (GC/FID). Isothermal separation at 1500 deg C on glass column packed with 10% SP-1000 on Chromosorb W-AW 100/120 mesh. The GC response of the samples was compared with that obtained from standard samples prepared by quantitative dilution of DBE in acetone to determine chamber concentration. The method permitted separation / identification of the 3 components to determine changes in composition

Duration of treatment / exposure:

98 days (males); 99 days (females)

Frequency of treatment:

5 days / week, 6 hours / day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/L air

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

0.16 mg/L air

Doses / concentrations 3

Dose / conc.:

0.4 mg/L air

Doses / concentrations 4

Dose / conc.:

1 mg/L air

No. of animals per sex per dose:

10 males, 10 females

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: Higest dose level selected regarded as maximum tolerated dose
- Rationale for animal assignment (if not random): Random

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 42/43 days and again after 92/93 days
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All study animals
- Parameters examined: Erythrocyte count; haemoglobin concentration; mean corpuscular volume; platelet count; leukocyte count including relative numbers of neutrophils, band neutrophils, lymphocytes, atypical lymphocytes, eosinophils, monocytes and basophils; haematocrit; mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 42/43 days and again after 92/93 days
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All study animals
- Parameters examined: Alkaline phosphatase; alanine aminotransferase; aspartate aminotransferase; glucose; urea nitrogen; calcium; cholesterol; creatinine; total protein; albumin; sodium; potassium; globulin

URINALYSIS: Yes
- Time schedule for collection of blood: 42/43 days and again after 92/93 days
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: .Volume; osmolality; pH; blood; sugar; protein; bilirubin; urobilinogen; ketones; colour; transparency; sediment

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes - Macroscopic examination and weighing of the brain, heart, lungs, liver, spleen, kidneys, testes and thymus. The following tissues were retained for microscopic examination: brain; spinal cord; peripheral nerve (sciatic); nasal cavity; larynx; trachea; lungs; heart; aorta; liver; pancreas; salivary glands; pharynx; oesophagus; stomach; duodenum; jejunum; ileum; caecum; colon; rectum; kidneys; urinary bladder; testes; epididymides; prostate; seminal vesicle; ovaries; uterus; cervix; vagina; pituitary; thyroid; parathyroid; adrenal; spleen; thymus; bone marrow (femur and sternum); mandibular lymph node; mesenteric lymph node; tracheobronchial lymph node; skeletal muscle; skin; mammary gland; eyes; harderian gland; exorbital lacrimal gland.


HISTOPATHOLOGY: Yes - Tissues listed above from all groups of male rats and from the control and high-dose female rats were examined microscopically. The nasal cavity from the low dose and intermediate-dose female rats were also examined.

Statistics:

One-way analysis of variance. When the F-test was significant, least significant difference (LSD) and Dunnett tests were used to compare data from the control group and treatment groups.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs for exposed animals were similar to those of control animals.
Animals exposed at 1.0 mg/L had wet fur during exposure due to aerosol deposition.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males and females of the l.0 mg/L exposure group exhibited depressed rates of weight gain compared to controls.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males in the high-dose group showed an increased haemoglobin at the end of the treatment period. At the same time, the absolute numbers of monocytes were decreased for the 0.16 mg/L and 1.0 mg/L exposure groups. Females exposed at 0.16 mg/L exhibited a decreased mean corpuscular volume both at the mid-point of the study and at the end of the treatment period.
These statistically significant differences were seen, values were within the range of biological variability or showed no dose-response relationship and were considered unrelated to treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A slight, but statistically significant, decrease in serum sodium concentration in males and females exposed at 1.0 mg/L at the midpoint of the study (42/43 days) and near the end of the 90-day exposure period. At the same time periods there was a slight, statistically significant, increase in serum calcium in female rats exposed at 0.40 and 1.0 mg/L.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the mid-point analyses, urine urobilinogen concentrations were increased in males of the 0.4 mg/L and 1.0 mg/L exposure groups. While statistically significant, these were within the range of biological variability or showed no dose-response relationship and were considered unrelated to treatment.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant and dose-related decrease in absolute and relative liver weights was seen in female rats in all treatment groups, relative to controls. In addition, males exposed at 1.0 mg/L exhibited a statistically significant decrease in absolute and relative liver weight.
Other statistically significant differences in the 1.0 mg/L exposure group included slight increases in relative heart and testes weights in males and a slight decrease in absolute spleen weight in female animals. These slight organ weight changes were not accompanied by any histopathological changes and are considered of minimal biological significance.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Squamous metaplasia was noted primarily in the olfactory epithelium in all treatment groups. The effect was minimal and seen in 3 of 20 rats exposed at 0.16 mg/L. The effect was of minimal to mild severity in 17/20 rats exposed at 0.40 mg/L, and 19/20 rats exposed at 1.0 mg/L groups. A single female in the 0.40 mg/L group showed lesions of moderate severity.
Other than these nasal lesions, examination showed no abnormal effects in exposed animals.

Details on results:

CLINICAL SIGNS AND MORTALITY: Clinical signs observed in substance exposed rats were similar to those seen in control animals. Animal exposed at 1.0 mg/L group had wet fur during exposure due to aerosol deposition, the fur drying within 2 hours after exposure ceased. One male rat of the 0.4 mg/L exposure group was killed after 35 days due to an accidentally fractured nasal septum.

BODY WEIGHT AND WEIGHT GAIN: Both males and females exposed at l.0 mg/L exhibited reduced body weight gain relative to controls.

HAEMATOLOGY: Apparent group differences seen in haematological measured parameters were regarded as being within the range of biological variability or showed no dose-response relationship and, as a result, were considered unrelated to treatment.

CLINICAL CHEMISTRY: There was a slight but statistically significant decrease in serum sodium concentration in males and females of the 1.0 mg/L exposure group at the midpoint of the study (day 42 for the males and day 43 for the females) and at the end of the study. There was a slight but statistically significant increase in serum calcium in female rats in the 0.40 and 1.0 mg/L exposure groups at the midpoint and at the end of the study.

URINALYSIS: Apparent group differences seen in those urine analysis parameters that wre measured were regarded as being within the range of biological variability or showed no dose-response relationship and were therefore considered unrelated to treatment.

ORGAN WEIGHTS: A statistically significant and dose-related reduction in both absolute and relative liver weights was seen in female rats in all treatment groups and in male rats exposed at 1.0 mg/L. Statistically significant differences between test and control animals were also apparent in animals exposed at 1.0 mg/L and included slight increases in relative heart and testes weights in males and a slight decrease in absolute spleen weight in females. These minor weight changes were not accompanied by any observed microscopic change in the tissue histopathological changes and were considered to be of minimal biological significance.

GROSS PATHOLOGY: No details available

HISTOPATHOLOGY: NON-NEOPLASTIC: Examination of the nasal areas revealed squamous metaplasia, primarily in the olfactory epithelium, in all treatment groups. The effect was minimal and noted in 3 of 20 rats exposed at 0.16 mg/L; in 17/20 animals exposed at 0.4 mg/L and in 19/20 animals exposed at 1.0 mg/L and was of minimal to mild severity. One female rat exposed at 0.40 mg/L showed lesions of moderate severity.

Effect levels

1

Dose descriptor:

LOAEC

Effect level:

0.16 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

other: histopathology: squamous metaplasia of nasal olfactory epithelium

2

Dose descriptor:

NOAEC

Effect level:

0.4 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Lack of significant systemic toxicity

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

0.16 mg/L air

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Applicant's summary and conclusion

Conclusions:

The mixture, at the concentrations examined, causes lesions of the olfactory epithelium. Other than this local effect, no significant systemic toxicity was apparent in the rat following repeated inhalation exposure over a period of at least 90 days.

Executive summary:

Groups of 10 male and 10 female rats have been exposed, by inhalation 6 hours/day/5 days/week, for approximately 14 weeks to a mixture of dibasic esters containing dimethyl succinate, dimethyl glutarate and dimethyl adipate. Animals were exposed at concentrations of 0.16, 0.40 or 1.0 mg/L.

 

Microscopic examination of the nasal areas revealed mild squamous metaplasia in the olfactory epithelium in all treated groups. Examination of other preserved tissues showed no abnormalities that could be attributed to treatment at any concentration tested. Other effects of exposure included a dose-dependent reduction in absolute and relative liver weights in female rats in all treatment groups and in male animals exposed at 1.0 mg/L. No microscopic changes were apparent in the liver and the biological significance of observed effects on liver weight is not known. A slight reduction in body weight and slightly reduced blood sodium concentrations was noted in males and females exposed at 1.0 mg/L. Slightly increased blood calcium concentrations were noted in females exposed at 0.40 or 1.0 mg/L. The slight changes in sodium and calcium concentrations were considered of minimal biological significance.

 

A NOAEC was not determined as a result of the squamous metaplasia observed in the rat olfactory epithelium after exposure to the mixture of dibasic esters - a local effect thought to be an irritant response to exposure. An exposure concentration of 0.4 mg/L may be regarded as a NOAEC for systemic toxicity.